Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

Bottom Line:
Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs).However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date.The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

ABSTRACTThe Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

fig_004: Comparison of amino acid sequences of the zinc-binding TRASH motifs inNpro. The amino acid sequences of the zinc-binding TRASH motifs in theNpro proteins of various BVDV strains deposited in the DDBJ/EMBL/GenBankdatabases were compared with those of GBK_E+ and GBK_E−. Atleast one strain per subgenotype [1a−1o (except for 1l) and 2a−2c]was chosen. The cysteines (Cs) at positions 112, 134 and 138 of the zinc-binding TRASHmotif are highlighted in bold and underlined. The amino acid residue at position 136is boxed in gray. The accession numbers of strains from the DDBJ/EMBL/GenBank are asfollows: NADL (M31182), SD-1 (M96751), Osloss (M96687), CP7 (U63479), Bega (AF049221),721 (AF144463), F (AF287284), 3186V6 (AF287282), W (AF287290), A (AF287283), G(AF287285), 23-15 (AF287279), KS86-1ncp (AB078950), SuwaNCP (KC853440), ZM-95(AF526381), Shitara/02/06 (AB359930), IS25CP/01 (AB359931), 890 (U18059),Hokudai-Lab/09 (AB567658) and NRW 14-13_Dup (−) (HG426485).

Mentions:
Comparison of amino acid sequences of zinc-binding TRASH motif inNpro: The amino acid sequences of the zinc-binding TRASH motifs in theNpro proteins from various BVDV strains deposited in the DDBJ/EMBL/GenBankdatabases were compared with those of the GBK_E+ and GBK_E− strains.At least one strain per subgenotype [1a−1o (except for 1l) and 2a−2c][11, 18] waschosen in the present study. As a result, BVDV strains, except for the G strain, containamino acid residues Cys112-Cys134-Asp136-Cys138 in the zinc-binding TRASH motif ofNpro, as observed in GBK_E+ (Fig.4Fig. 4.

Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

fig_004: Comparison of amino acid sequences of the zinc-binding TRASH motifs inNpro. The amino acid sequences of the zinc-binding TRASH motifs in theNpro proteins of various BVDV strains deposited in the DDBJ/EMBL/GenBankdatabases were compared with those of GBK_E+ and GBK_E−. Atleast one strain per subgenotype [1a−1o (except for 1l) and 2a−2c]was chosen. The cysteines (Cs) at positions 112, 134 and 138 of the zinc-binding TRASHmotif are highlighted in bold and underlined. The amino acid residue at position 136is boxed in gray. The accession numbers of strains from the DDBJ/EMBL/GenBank are asfollows: NADL (M31182), SD-1 (M96751), Osloss (M96687), CP7 (U63479), Bega (AF049221),721 (AF144463), F (AF287284), 3186V6 (AF287282), W (AF287290), A (AF287283), G(AF287285), 23-15 (AF287279), KS86-1ncp (AB078950), SuwaNCP (KC853440), ZM-95(AF526381), Shitara/02/06 (AB359930), IS25CP/01 (AB359931), 890 (U18059),Hokudai-Lab/09 (AB567658) and NRW 14-13_Dup (−) (HG426485).

Mentions:
Comparison of amino acid sequences of zinc-binding TRASH motif inNpro: The amino acid sequences of the zinc-binding TRASH motifs in theNpro proteins from various BVDV strains deposited in the DDBJ/EMBL/GenBankdatabases were compared with those of the GBK_E+ and GBK_E− strains.At least one strain per subgenotype [1a−1o (except for 1l) and 2a−2c][11, 18] waschosen in the present study. As a result, BVDV strains, except for the G strain, containamino acid residues Cys112-Cys134-Asp136-Cys138 in the zinc-binding TRASH motif ofNpro, as observed in GBK_E+ (Fig.4Fig. 4.

Bottom Line:
Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs).However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date.The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

ABSTRACTThe Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.